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Opt Express. 2010 Nov 8;18(23):23924-32. doi: 10.1364/OE.18.023924.

Label-free optical imaging of membrane patches for atomic force microscopy.

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JILA, National Institute of Standards and Technology and Department of Physics, University of Colorado, Boulder, Colorado 80309, USA.


In atomic force microscopy (AFM), finding sparsely distributed regions of interest can be difficult and time-consuming. Typically, the tip is scanned until the desired object is located. This process can mechanically or chemically degrade the tip, as well as damage fragile biological samples. Protein assemblies can be detected using the back-scattered light from a focused laser beam. We previously used back-scattered light from a pair of laser foci to stabilize an AFM. In the present work, we integrate these techniques to optically image patches of purple membranes prior to AFM investigation. These rapidly acquired optical images were aligned to the subsequent AFM images to ~40 nm, since the tip position was aligned to the optical axis of the imaging laser. Thus, this label-free imaging efficiently locates sparsely distributed protein assemblies for subsequent AFM study while simultaneously minimizing degradation of the tip and the sample.

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